Derivatization of oligosaccharides

ABSTRACT

A method for purifying, separating and/or isolating an oligosaccharide or a salt thereof is presented. An embodiment of the invention is based upon the formation of anomeric O-benzyl/substituted O-benzyl derivatives in a selective anomeric alkylation reaction.

In the present years manufacture and commercialization of complexcarbohydrates including secreted oligosaccharides have increasedsignificantly due to their roles in numerous biological processesoccurring in living organisms. Secreted oligosaccharides such as humanmilk oligosaccharides (HMOs) are carbohydrates which have gained muchinterest in recent years and are becoming important commercial targetsfor nutrition and therapeutic industries. In particular the synthesis ofthese HMOs has increased significantly due to the role of HMOs innumerous biological processes occurring in humans. The great importanceof HMOs is directly linked to their unique biological activities such asantibacterial, antiviral, immune system and cognitive developmentenhancing activities. Human milk oligosaccharides are found to act asprebiotics in the human intestinal system helping to develop andmaintain the intestinal flora. Furthermore they have also proved to beanti-inflammatory, and therefore these compounds are attractivecomponents in the nutritional industry for the production of, forexample, infant formulas, infant cereals, clinical infant nutritionalproducts, toddler formulas, or as dietary supplements or healthfunctional food for children, adults, elderly or lactating women, bothas synthetically composed and naturally occurring compounds and saltsthereof. Likewise, the compounds are also of interest in the medicinalindustry for the production of therapeutics due to their prognostic useas immunomodulators. However, the syntheses and purification of theseoligosaccharides and their intermediates remained a challenging task forscience.

To date, access to large volumes of human milk oligosaccharides has notbeen possible by using isolation, biotechnology and syntheticmethodologies.

The availability of naturally occurring sialylated human milkoligosaccharides is limited from natural sources. Mature human milk isthe natural milk source that contains the highest concentrations of milkoligosaccharides (12-14 g/l), other milk sources are cow's milk (0.01g/l), goat's milk and milk from other mammals. Approximately 200 HMOshave been detected from human milk by means of combination of techniquesincluding microchip liquid chromatography mass spectrometry (HPLCChip/MS) and matrix-assisted laser desorption/ionization Fouriertransform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS)(Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which todate at least 115 oligosaccharides have been structurally determined(Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011).These human milk oligosaccharides can be grouped into 13 core units(Table 1). Due to the large number of similar HMOs and their lowconcentrations in mammalian milk, isolation of HMOs is a difficult taskeven in milligram quantities. To date only analytical HPLC methodologieshave been developed for the isolation of some HMOs from natural sources.It is therefore difficult to provide suitable HMO replacements in foods,particularly in infant formulae which display at least part of theentire spectrum of HMOs.

TABLE 1 13 different core structures of human milk oligosaccharides NoCore name Core structure 1 lactose (Lac) Galβ1-4Glc 2 lacto-N-tetraose(LNT) Galβ1-3GlcNAcβ1-3Galβ1-4Glc 3 lacto-N-neotetraoseGalβ1-4GlcNAcβ1-3Galβ1-4Glc (LNnT) 4 lacto-N-hexaose (LNH)Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc 5 lacto-N-neohexaoseGalβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc (LNnH) 6para-lacto-N-hexaose Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc(para-LNH) 7 para-lacto-N-neohexaoseGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc (para-LNnH) 8lacto-N-octaose (LNO) Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc 9 lacto-N-neooctaoseGalβ1-4GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1- (LNnO) 4GlcNAcβ1-6)Galβ1-4Glc10 Iso-lacto-N-octaose (iso- Galβ1-3GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-LNO) 4GlcNAcβ1-6)Galβ1-4Glc 11 para-lacto-N-octaoseGalβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1- (para-LNO)4GlcNAcβ1-3Galβ1-4Glc 12 Lacto-N-neodecaoseGalβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-3(Galβ1- (LNnD)4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc 13 Lacto-N-decaose (LND)Galβ1-3GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc

The chemical synthesis of human milk oligosaccharides is one of the mostchallenging fields of carbohydrate chemistry due to the nature of theglycosyl donors, especially in the case of the manufacture of sialylHMOs. Careful selection of a glycosyl donor-acceptor match, kinetic andsolvent effects is always needed in synthetic methodologies, which infact comprise numerous reaction steps, protecting group manipulationsand chromatographic purification, poor yields, and provide onlymilligram quantities of HMOs. Thus, these methods do not offerattractive techniques for large scale preparation.

In case of enzymatic production of HMOs, glycosyltransferases andglycosidases are the preferred enzymes used. These complex enzymaticsystems represent very expensive methodologies for scale up productions.The use of glycosidases is often characterized by poor yield andmoderate regio- and/or stereoselectivity which may cause difficultpurification problems. This prevents its use in industrial scaletechnology developments. Glycosyltransferases require the presence ofnucleotide type glycosyl donors, the availability of which is ratherlimited.

Some biotechnological methodologies are also described using geneticallymodified bacteria, yeast or other microorganisms. Such methodologieshave serious drawbacks in regulatory processes.

In summary, isolation technologies have never been able to provide largequantities of human milk oligosaccharides due to the large number ofoligosaccharides present in human milk. Additionally, the presence ofregioisomers characterized by extremely similar structures further madeseparation technologies unsuccessful. Enzymatic methodologies sufferfrom such problems as the low availability of enzymes, extremely highsugar nucleotide donor prices and regulatory difficulties due to the useof enzymes produced in genetically modified organisms. The preparationof human milk oligosaccharides via biotechnology has huge regulatoryobstacles due to the potential formation of several unnaturalglycosylation products. To date, the chemical methods developed for thesynthesis of HMOs have several drawbacks which prevented the preparationof even multigram quantities. The most severe drawback of chemicalapproaches is the lack of design for crystalline intermediates tofacilitate low cost purification methodologies and to enhance scale-upopportunities.

There is still a need for novel methodologies which can simplifypreparation and overcome or avoid purification problems encountered inprior art methods. In addition, there is an urgent need in the art forproviding complex oligosaccharides and mixtures thereof, which resembleas good as possible or even imitate the variety of complexoligosaccharides in human milk.

The present invention provides a general derivatization method, theresult of which are HMO derivatives having beneficial features forovercoming isolation and/or purification problems characterized by theprior art.

Some individual HMO derivatives obtainable by the inventive method to bespecified later are known in the prior art: 1-O-β-benzyl-LNnT (Ponpipomet al. Tetrahedron Lett. 20, 1717 (1978)),1-O-β-(4-hydroxymethylbenzyl)-LNnT (Yan et al. Carbohydr. Res. 328, 3(2000)), 1-O-β-benzyl-LNT (Malleron et al. Carbohydr. Res. 341, 29(2006), Liu et al. Bioorg. Med. Chem. 17, 4910 (2009)),1-O-β-benzyl-6′-O-sialyl-lactose Na salt (Rencurosi et al. Carbohydr.Res. 337, 473 (2002)), 1-O-β-benzyl-3′-O-sialyl-lactose Na salt(Rencurosi et al. Carbohydr. Res. 337, 473 (2002), WO 96/32492 A2),1-O-β-(4,5-dimethoxy-2-nitro)-benzyl-3′-O-sialyl-lactose Na salt (Cohenet al. J. Org. Chem. 65, 6145 (2000)).

Whatever route is taken to synthesise or isolate an oligosaccharide, thefinal target unprotected oligosaccharide is soluble only in water, whichpresents challenges for the later steps of the synthesis or forisolation/separation/purification methods. Organic solvents commonlyused in synthetic manufacturing processes are not suitable for thereactions of the very final stages of the oligosaccharide synthesis.Additionally, the presence of numbers of similar compounds either innatural source or obtained in enzyme catalyzed syntheses likewisedemands powerful chromatographic system having polar aqueous milieuwhich is difficult to find.

The present invention provides methodology suitable for derivatizingHMOs. The invention is based upon the formation of anomericO-benzyl/substituted O-benzyl derivatives in a selective anomericalkylation reaction.

Accordingly, the present invention relates to a method for purifying,separating and/or isolating an oligosaccharide of general formula 1 or asalt thereof

-   -   wherein R₁ is fucosyl or H,    -   R₂ is fucosyl or H,    -   R₃ is selected from H, sialyl, N-acetyl-lactosaminyl and        lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group        may carry a glycosyl residue comprising one or more        N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups;        each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can        be substituted with one or more sialyl and/or fucosyl residue,    -   R₄ is selected from H, or sialyl and N-acetyl-lactosaminyl        groups optionally substituted with a glycosyl residue comprising        one or more N-acetyl-lactosaminyl and/or one or more        lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and        lacto-N-biosyl groups can be substituted with one or more sialyl        and/or fucosyl residue,    -   wherein at least one of the R₁, R₂, R₃ or R₄ groups differs from        H,        comprising the steps:        a) one or more compounds of general formula 1 is/are subjected        to an anomeric O-alkylation reaction in the presence of R—X to        yield a mixture comprising one or more compounds of general        formula 2 or salts thereof

-   -   wherein X is a leaving group such as halogen, alkyl- or        arylsulfonyloxy, R is a group removable by hydrogenolysis, and        R₁, R₂, R₃ and R₄ are as defined above, and wherein at least one        of the R₁, R₂, R₃ or R₄ groups differs from H,        b) the mixture comprising one or more compounds of general        formula 2 obtained in step a) is subjected to chromatography        and/or crystallization to give one or more individual compounds        of general formula 2 each in substantially pure form,        c) an individual compound of general formula 2 in substantially        pure form obtained in step b) is subjected to catalytic        hydrogenolysis to yield a compound of general formula 1.

Throughout the present application the term “protecting group that isremovable by hydrogenolysis” or “group removable by hydrogenolysis”refers to groups whose C—O bond to the 1-oxygen can be cleaved byaddition of hydrogen in the presence of catalytic amounts of palladium,Raney nickel or another appropriate metal catalyst known for use inhydrogenolysis, resulting in the regeneration of the OH group. Suchprotecting groups are well known to the skilled man and are discussed inProtective Groups in Organic Synthesis, P G M Wuts and T W Greene, JohnWiley & Sons 2007. Suitable protecting groups include benzyl,diphenylmethyl(benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl ortriphenylmethyl(trityl) groups, each of which may be optionallysubstituted by one or more groups selected from: alkyl, alkoxy, phenyl,amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl,alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl,azido, halogenalkyl or halogen. Preferably, such substitution, ifpresent, is on the aromatic ring(s). Particularly preferred protectinggroups are benzyl or 1- or 2-naphthylmethyl groups optionallysubstituted with one or more groups selected from phenyl, alkyl orhalogen. More preferably, the protecting group is selected fromunsubstituted benzyl, unsubstituted 1-naphthylmethyl, unsubstituted2-naphthylmethyl, 4-chlorobenzyl, 3-phenylbenzyl, 4-methylbenzyl and4-nitrobenzyl.

“Compound in substantially pure form”, when referring to a compound ofgeneral formula 2, means that the compound contains less than 5 w/w % ofimpurities, preferably less than 3 w/w % of impurities, more preferablyless than 1 w/w % of impurities, most preferably less than 0.5 w/w % ofimpurities, in particular less than 0.1 w/w % of impurities, wherein“impurities” refers to any physical entity different to that compound,such as unreacted intermediate(s) remaining from the synthesis of thecompound of general formula 2, by-product(s), degradation product(s),inorganic salt(s) and/or other contaminations other than organicsolvent(s) and/or water.

Throughout the present description, the term “alkyl” means a linear orbranched chain saturated hydrocarbon group with 1-6 carbon atoms, suchas methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl,t-butyl, n-hexyl, etc.

The term “aryl” refers to a homoaromatic group such as phenyl ornaphthyl.

In the present description, the term “acyl” represents anR′—C(═O)-group, wherein R′ may be H, alkyl (see above) or aryl (seeabove), such as formyl, acetyl, propionyl, butyryl, pivaloyl, benzoyl,etc. The alkyl or aryl residue may either be unsubstituted or may besubstituted with one or more groups selected from alkyl (only for arylresidues), halogen, nitro, aryl, alkoxy, amino, alkylamino,dialkylamino, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl,N,N-dialkylcarbamoyl, azido, halogenalkyl or hydroxyalkyl, giving riseto acyl groups such as chloroacetyl, trichloroacetyl, 4-chlorobenzoyl,4-nitrobenzoyl, 4-phenylbenzoyl, 4-benzamidobenzoyl,4-(phenylcarbamoyl)-benzoyl etc.

The term “alkyloxy” or “alkoxy” means an alkyl group (see above)attached to the parent molecular moiety through an oxygen atom, such asmethoxy, ethoxy, t-butoxy, etc.

“Halogen” means fluoro, chloro, bromo or iodo.

“Amino” refers to a —NH₂ group.

“Alkylamino” means an alkyl group (see above) attached to the parentmolecular moiety through an —NH-group, such as methylamino, ethylamino,etc.

“Dialkylamino” means two alkyl groups (see above), either identical ordifferent ones, attached to the parent molecular moiety through anitrogen atom, such as dimethylamino, diethylamino, etc.

“Acylamino” refers to an acyl group (see above) attached to the parentmolecular moiety through an —NH-group, such as acetylamino (acetamido),benzoylamino (benzamido), etc.

“Carboxyl” denotes an —COOH group.

“Alkyloxycarbonyl” means an alkyloxy group (see above) attached to theparent molecular moiety through a —C(═O)-group, such as methoxycarbonyl,t-butoxycarbonyl, etc.

“Carbamoyl” is an H₂N—C(═O)-group.

“N-Alkylcarbamoyl” means an alkyl group (see above) attached to theparent molecular moiety through a —HN—C(═O)-group, such asN-methylcarbamoyl, etc.

“N,N-Dialkylcarbamoyl” means two alkyl groups (see above), eitheridentical or different ones, attached to the parent molecular moietythrough a >N—C(═O)-group, such as N,N-methylcarbamoyl, etc.

In the present description the term “salt” in connection with compoundsof general formulae 1 and 2, which contain at least one sialyl residue,means an associated ion pair consisting of the negatively charged acidresidue and one or more cations in any stoichiometric proportion.Cations, as used in the present context are atoms or molecules withpositive charge. The cation may be inorganic as well as organic cation.Preferred inorganic cations are ammonium ion, alkali metal, alkali earthmetal and transition metal ions, more preferably Na⁺, K⁺, Ca²⁺, Mg²⁺,Ba²⁺, Fe²⁺, Zn²⁺, Mn²⁺ and Cu²⁺, most preferably K⁺, Ca²⁺, Mg²⁺, Ba²⁺,Fe²⁺ and Zn²⁺. Basic organic compounds in positively charged form may berelevant organic cations. Such preferred positively charged counterpartsare diethyl amine, triethyl amine, diisopropyl ethyl amine,ethanolamine, diethanolamine, triethanolamine, imidazol, piperidine,piperazine, morpholin, benzyl amine, ethylene diamine, meglumin,pyrrolidine, choline, tris-(hydroxymethyl)-methyl amine,N-(2-hydroxyethyl)-pyrrolidine, N-(2-hydroxyethyl)-piperidine,N-(2-hydroxyethyl)-piperazine, N-(2-hydroxyethyl)-morpholine,L-arginine, L-lysine, oligopeptides having L-arginine or L-lysine unitor oligopeptides having free amino group on N-terminal, etc., all inprotonated form. Such salt formations can be used to modifycharacteristics of the complex molecule as a whole, such as stability,compatibility to excipients, solubility and ability to form crystals.

Furthermore, the term “fucosyl” within the context of the presentinvention means a L-fucopyranosyl group attached to the coreoligosaccharide with α-interglycosidic linkage:

“N-acetyl-lactosaminyl” group within the context of the presentinvention means the glycosyl residue of N-acetyl-lactosamine (LacNAc,Galpβ1-4GlcNAcp) linked with β-linkage:

Furthermore, the term “Lacto-N-biosyl” group within the context of thepresent invention means the glycosyl residue of lacto-N-biose (LNB,Galpβ1-3GlcNAcp) linked with β-linkage:

The term “sialyl” within the context of the present invention means theglycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac)linked with α-linkage:

Additionally, the term “glycosyl residue comprising one or moreN-acetyl-lactosaminyl and/or one or more lacto-N-biosyl units” withinthe context of the present invention preferably means a linear orbranched structure comprising the said units that are linked to eachother by interglycosidic linkages.

The term “anomeric O-alkylation” in the present context means theselective alkylation of the anomeric OH group in the presence ofnon-protected primary and secondary OHs of the starting compound.Particularly, two basic methodologies shall be describedwith respect tothe anomeric O-alkylation used in the present invention. When a compoundof general formula 1 is devoid of any sialyl residues (neutraloligosaccharides) the alkylation reaction is performed in a dipolaraprotic solvent such as DMF, DMSO, N-methylpyrrolidone,hexamethylphosphoramide (HMPA), N,N′-dimethylhexahydropyrimidine-2-one(DMPU), THF, dioxane, acetonitrile, etc., or mixture thereof, in thepresence of a strong base and R—X wherein X is a leaving group selectedfrom halogen, alkylsulfonyloxy like mesyl, triflyl, etc. andarylsulfonyl like benzenesulfonyl, tosyl, etc. Preferred alkylatingagents are benzyl or f- or 2-naphthylmethyl halogenides optionallysubstituted with one or more groups selected from phenyl, alkyl orhalogen. The strong base is able to deprotonate the anomeric OHchemoselectively due to its more acidic character when an equivalentamount or a slight excess (1 to 1.5 equiv.) of base is used. The strongbase suitable for activating the anomeric OH is typically taken from thegroup of alkali metal or alkaline earth metal hydrides or alkoxides suchas NaH, KH, CaH₂, NaOMe, NaO^(t)Bu, KO^(t)Bu, inorganic hydroxides,potassium carbonate, etc. The alkylation agent is added in an equivalentamount or a slight excess (1 to 1.5 equiv.). The reaction is carried outbetween −10 and 80° C., preferably at a low temperature during wholecourse of the reaction or at a low temperature during the addition ofthe reagents/reactants and an elevated temperature in the later stagesof the course of the reaction. Neutral oligosaccharidebenzyl/substituted benzyl glycosides of general formula 2 can beobtained after usual work-up. In case of acidic oligosaccharides, thatis, wherein at least one sialyl residue is present, the cesium salt ofthe starting material, previously formed by treating the acidic compoundwith cesium carbonate, is used for masking the carboxylate group inmethyl ester form before anomeric O-alkylation. To form the methylester, the cesium salt is dissolved in a dipolar aprotic solvent such asDMF, DMSO, N-methylpyrrolidone, hexamethylphosphoramide (HIMPA),N,N′-dimethylhexahydropyrimidine-2-one (DMPU), THF, dioxane,acetonitrile, etc., or mixture thereof, and a methylating agent likemethyl iodide, methyl triflate, dimethyl sulphate or the like is addedin equivalent amount or slight excess (1 to 1.5 equiv.) with respect tothe cesium salt. The reaction typically takes place in 4-24 hours. Theresulting methyl ester compound is then subjected to anomericO-alkylation as specified above. At the end of the reaction the mixtureis diluted with water giving rise to a basic aqueous conditions underwhich the methyl ester is cleaved resulting in the formation of anacidic oligosaccharide benzyl/substituted benzyl glycoside of generalformula 2.

In step a) a compound of general formula 1 is generally available as acrude product accompanied by unreacted precursors, reagents, by-productsand other contaminants from the chemical, enzymatic or chemo-enzymaticsynthesis of said compound. Where compounds of general formula 1 areobtained from a natural source, they may be contaminated by organicmolecules such as amino acids, oligo- and polypeptides, lipids, lactose,monosaccharides, vitamins, etc., which contamination profile may becharacteristic to the natural pool from which the compounds of generalformula 1 were obtained.

In step b) the crude mixture comprising one or more compounds of generalformula 2 obtained in step a), accompanied by the contaminants mentionedabove and derivatives thereof as well as traces of reagents used in theanomeric O-alkylation, is separated by chromatography and/orcrystallization to give one or more individual compounds of generalformula 2 each in substantially pure form. That is, where more than onecompound of general formula 2 is obtained in step (b), each of thosecompounds is obtained separately from and substantially free of any ofthe other compounds of general formula 2 also obtained in that step. Thechromatographic means can be any suitable separation techniques such ascolumn chromatography, HPLC, reverse phase chromatography, sizeexclusion chromatography, or ion exchange chromatography. generally usedfor the separation, isolation and/or purification of carbohydrates.

In step c) “catalytic hydrogenolysis” means the removal of the R-groupwhich typically takes place in a protic solvent or in a mixture ofprotic solvents. Step (c) is conducted on a single compound of generalformula 2 obtained in step (b). A protic solvent may be selected fromthe group consisting of water, acetic acid or C₁-C₆ alcohol. A mixtureof one or more protic solvents with one or more suitable aprotic organicsolvents miscible partially or fully with the protic solvent(s) (such asTHF, dioxane, ethyl acetate, acetone, etc.) may also be used. Water, oneor more C₁-C₆ alcohols or a mixture of water and one or more C₁-C₆alcohols are preferably used as the solvent system. The solutionscontaining the carbohydrate derivatives may have any suitableconcentration, and suspensions of the carbohydrate derivatives with theselected solvent(s) may also be used. The reaction mixture is stirred at10-100° C. temperature range, preferably between 20-70° C., in ahydrogen atmosphere of 1-50 bar in the presence of a catalyst such aspalladium, Raney nickel or any other appropriate metal catalyst,preferably palladium on charcoal or palladium black, until reaching thecompletion of the reaction. Catalyst metal concentrations generallyrange from 0.1% to 10% based on the weight of carbohydrate. Preferably,the catalyst concentrations range from 0.15% to 5%, more preferably0.25% to 2.25%. Transfer hydrogenation may also be performed, when thehydrogen is generated in situ from cyclohexene, cyclohexadiene, formicacid or ammonium formate. Addition of organic or inorganic bases oracids and/or basic or acidic ion exchange resins can also be used toimprove the kinetics of the hydrogenolysis. The use of basic substancesis especially preferred when halogen substituents are present on thesubstituted benzyl moieties of the precursors. Preferred organic basesinclude but are not limited to triethylamine, diisopropyl ethylamine,ammonia, ammonium carbamate, diethylamine, etc. Preferredorganic/inorganic acids include but are not limited to formic acid,acetic acid, propionic acid, chloroacetic acid, dichloroacetic acid,trifluoroacetic acid, HCl, HBr, etc. The conditions proposed above allowsimple, convenient and delicate removal of the solvent(s) giving rise topure compound of general formula 1. The compound of general formula 1can be isolated from the reaction mixture using conventional work-upprocedures in crystalline, amorphous solid, syrupy form or concentratedaqueous solution.

It should be emphasized that the introduction of the R-group bringsnumerous advantageous features to compounds of general formula 2 withrespect to the isolation, separation and/or purification of a compoundof general formula 2. Firstly, the R-group as an apolar moiety changesthe polarity of the entire molecule, and thus it enlarges the repertoireof column packings and elution systems that a skilled person hasavailable for selecting the best suitable conditions in order to achievethe best result. For example, due to the more different polarity of thecompounds present a reverse phase chromatographic separation could beeasily performed when water is used, as compounds of general formula 2migrate much more slowly than the very polar compounds present in thereaction mixture, thus the polar compounds can be eluted smoothly.Compounds of general formula 2 can be then washed from the column withe.g. alcohol. Secondly, R-groups are aromatic moieties, and thus canserve as chromophores offering the possibility of UV-detection whicheases the identification of the desired objects. Thirdly, with carefulselection of the R-groups crystalline materials of general formula 2 maybe obtained. Crystallization or recrystallization is one of the simplestand cheapest methods to isolate a product from a reaction mixture,separate it from contaminations and obtain pure substance. Isolation orpurification that uses crystallization makes the whole technologicalprocess robust and cost-effective, and thus it is advantageous andattractive compared to other procedures. Fourthly, removal of theR-group from a compound of general formula 2 takes place under delicateconditions nearly quantitatively without the threat of by-productformation. R-groups such as benzyl/substituted benzyl protective groupsare converted exclusively into toluene/substituted toluene underhydrogenolysis conditions and they can easily be removed even on a multiton scale from water soluble oligosaccharide products via evaporationand/or extraction processes. Thus the chemical/stereochemical purity ofa compound of general formula 1 is directly linked to that of a compoundof general formula 2.

In a preferred method a crude mixture comprising one or more compoundsof general formulae 1a, 1b or 1c, or salts of these compounds, all ofwhich fall within the scope of compounds of general formula 1,

is subjected to derivatization in step a) to give a crude mixturecomprising one or more compounds of general formulae 2a, 2b or 2c, orsalts of these compounds, all of which fall within the scope ofcompounds of general formula 2, respectively,

-   -   wherein R, R₁ and R₂ are as defined above    -   R_(3a) is an N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one N-acetyl-lactosaminyl        and/or one lacto-N-biosyl group; each of the        N-acetyl-lactosaminyl and lacto-N-biosyl groups can be        substituted with one or more sialyl and/or fucosyl residue,    -   R_(4a) is H or an N-acetyl-lactosaminyl group optionally        substituted with a lacto-N-biosyl group; each of the        N-acetyl-lactosaminyl and lacto-N-biosyl groups can be        substituted with one or more sialyl and/or fucosyl residue,    -   R_(3b) is a lacto-N-biosyl group optionally substituted with one        or more sialyl and/or fucosyl residue(s),    -   R_(4b) is H or an N-acetyl-lactosaminyl group optionally        substituted with one or two N-acetyl-lactosaminyl and/or one        lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and        lacto-N-biosyl groups can be substituted with one or more sialyl        and/or fucosyl residues,    -   R₅ is independently H or sialyl,        wherein at least one of R₁, R₂ or R₅ differs from H,        the individual compounds of general formulae 2a, 2b or 2c are        isolated in substantially pure form by means of chromatography        and/or crystallization in step b) and an individual compound of        general formulae 2a, 2b or 2c converted into a compound of        general formulae 1a, 1b or 1c in step c).

Particularly preferably, compounds used and obtained according to theinventive method as defined above are characterized by their linkagesand modifications. Preferably, the compounds used and obtained in stepa) of the inventive method and obtained in step c) as defined accordingto general formulae 1a, 1b, 2a or 2b are characterized in that:

-   -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(3a) in general formulae 1a or 2a is attached to another        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(3a) in        general formula 1a or 2a is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(4a) in        general formula 1a or 2a is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(4b) in general formula 1b or 2b is attached to another        N-acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic        linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(4b) in        general formula 1b or 2b is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage.

Preferably, the compounds involved in the inventive method arecharacterized in that general formula 1a or 2a representslacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose,lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaosederivatives optionally substituted with one or more sialyl and/orfucosyl residue, or salts of these compounds, and general formula 1b or2b represents lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose,iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose derivativesoptionally substituted with one or more sialyl and/or fucosyl residue,or salts of these compounds.

More preferably, the compounds participating in the inventive methodspecified above are characterized in that:

-   -   the fucosyl residue attached to the N-acetyl-lactosaminyl and/or        the lacto-N-biosyl group is linked to        -   the galactose of the lacto-N-biosyl group with a 1-2            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            1-4 interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group            with a 1-3 interglycosidic linkage,    -   the sialyl residue attached to the N-acetyl-lactosaminyl and/or        the lacto-N-biosyl group is linked to        -   the galactose of the lacto-N-biosyl group with a 2-3            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            2-6 interglycosidic linkage and/or        -   the galactose of the N-acetyl-lactosaminyl group with a 2-6            interglycosidic linkage.

Most preferably, the following human milk oligosaccharides may bederivatized by the claimed method: 2′-O-fucosyllactose,3-O-fucosyllactose, 2′,3-di-O-fucosyllactose, 3′-O-sialyllactose,6′-O-sialyllactose, 3′-O-sialyl-3-O-fucosyllactose, lacto-N-tetraose,lacto-N-neotetraose, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNFP-I),Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (LNFP-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc (LNFP-III),Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-4)Glc (LNFP-V),Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LST-a),Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (LST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LST-c),Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FLST-a),Fucα1-2Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (FLST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FLST-c), Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-I), Galβ1-3(Fucα1-4)GlcNAcβ1-3 Galβ1-4(Fucα1-3)Glc (LNDFH-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-III),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (DS-LNT),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FDS-LNT I)and Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FDS-LNTII), or salts thereof. The R-glycosides may be alpha- or beta-anomers.Preferably, said R-glycosides are the beta-anomers.

The second aspect of the present invention provides benzyl orsubstituted benzyl glycosides of human milk oligosaccharides oranalogues. These can be prepared from the crude reaction mixturecomprising one or more HMOs or analogues thereof characterized bygeneral formula 1 according to step a) of the claimed method asspecified above. Thus the present invention provides compounds ofgeneral formula 2′ or salts thereof

-   -   wherein R is a group removable by hydrogenolysis,    -   R₁ is fucosyl or H,    -   R₂ is fucosyl or H,    -   R₃ is selected from H, sialyl, N-acetyl-lactosaminyl and        lacto-N-biosyl groups,    -   wherein the N-acetyl lactosaminyl group may carry a glycosyl        residue comprising one or more N-acetyl-lactosaminyl and/or one        or more lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl        and lacto-N-biosyl groups can be substituted with one or more        sialyl and/or fucosyl residue,    -   R₄ is selected from H, sialyl and N-acetyl-lactosaminyl groups        optionally substituted with a glycosyl residue comprising one or        more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl        groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl        groups can be substituted with one or more sialyl and/or fucosyl        residue,        wherein at least one of the R₁, R₂, R₃ or R₄ groups differs from        H, and provided that the following compounds are excluded:        R-glycosides of LNnT, 1-O-β-benzyl-LNT, R-glycosides of        6′-O-sialyl-lactose and salts thereof,        1-O-β-benzyl-3′-O-sialyl-lactose Na salt,        1-O-β-(4,5-dimethoxy-2-nitro)-benzyl-3′-O-sialyl-lactose Na        salt.

In a preferred embodiment compounds of general formula 2′ arecharacterized by general formulae 2′ a, 2′ b or 2′ c or salts thereof

-   -   wherein R, R₁ and R₂ are as defined above    -   R_(3a) is an N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one N-acetyl-lactosaminyl        and/or one lacto-N-biosyl group; each of the        N-acetyl-lactosaminyl and lacto-N-biosyl groups can be        substituted with one or more sialyl and/or fucosyl residue,    -   R_(4a) is H or an N-acetyl-lactosaminyl group optionally        substituted with a lacto-N-biosyl group; each of the        N-acetyl-lactosaminyl and lacto-N-biosyl groups can be        substituted with one or more sialyl and/or fucosyl residue,    -   R_(3b) is a lacto-N-biosyl group optionally substituted with one        or more sialyl and/or fucosyl residue,    -   R_(4b) is H or an N-acetyl-lactosaminyl group optionally        substituted with one or two N-acetyl-lactosaminyl and/or one        lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and        lacto-N-biosyl groups can be substituted with one or more sialyl        and/or fucosyl residue,    -   R₅ is independently H or sialyl,        wherein at least one of R₁, R₂ or R₅ differs from H.

Particularly preferably, compounds according general formulae 2′ a or 2′b as defined above are further characterized by their linkages andmodifications. Preferably, the compounds as defined according to generalformulae 2′ a or 2′ b are characterized in that:

-   -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(3a) in general formulae 1′ a or 2′a is attached to another        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(3a) in        general formula 1′ a or 2′a is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(4a) in        general formula 1′ a or 2′a is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage,    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(4b) in general formula I′ a or 1′ b is attached to another        N-acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycosidic        linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(4b) in        general formula 1′a or 1′b is attached to the        N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage.

Preferably, the compounds involved in the inventive method arecharacterized in that general formula 2′ a representslacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose,lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaoseR-glycosides optionally substituted with one or more sialyl and/orfucosyl residue, or salts thereof, and general formula 2′ b representslacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose,lacto-N-decaose and lacto-N-neodecaose R-glycosides optionallysubstituted with one or more sialyl and/or fucosyl residue, or saltsthereof.

More preferably, the compounds participating specified above are furthercharacterized in that:

-   -   the fucosyl residue attached to the N-acetyl-lactosaminyl and/or        the lacto-N-biosyl group is linked to        -   the galactose of the lacto-N-biosyl group with a 1-2            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            1-4 interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group            with a 1-3 interglycosidic linkage,    -   the sialyl residue attached to the N-acetyl-lactosaminyl and/or        the lacto-N-biosyl group is linked to        -   the galactose of the lacto-N-biosyl group with a 2-3            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            2-6 interglycosidic linkage and/or        -   the galactose of the N-acetyl-lactosaminyl group with a 2-6            interglycosidic linkage.

Most preferably, the R-glycosides of the following human milkoligosaccharides are provided: 2′-O-fucosyllactose, 3-O-fucosyllactose,2′,3-di-O-fucosyllactose, 3′-O-sialyllactose, 6′-O-sialyllactose,3′-O-sialyl-3-O-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose,Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNFP-I),Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (LNFP-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc (LNFP-III),Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-4)Glc (LNFP-V),Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LST-a),Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (LST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LST-c),Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FLST-a),Fucα1-2Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (FLST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3 Galβ1-4(Fucα1-3)Glc (FLST-c), Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3 Galβ1-4(Fucα1-3)Glc (LNDFH-I), Galβ1-3(Fucα1-4)GlcNAcβ1-3 Galβ1-4(Fucα1-3)Glc (LNDFH-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-III),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (DS-LNT),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FDS-LNT I)and Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FDS-LNTII), or salts thereof.

It is strongly emphasised that compounds characterized by generalformula 2′ can be considered as sole chemical entities such as either aor β anomers or even an anomeric mixture of α and β isomers, preferablyas the β-anomer. Compounds of general formula 2′ can be characterized ascrystalline solids, oils, syrups, precipitated amorphous material orspray dried products. If crystalline, compounds of general formula 2′might exist either in anhydrous or in hydrated crystalline forms byincorporating one or several molecules of water into their crystalstructures. Similarly, compounds characterized by general formula 2′might exist as crystalline substances incorporating ligands such asorganic molecules and/or ions into their crystal structures.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not to be limiting thereof.

EXAMPLES

-   -   1. General procedure for the preparation of compounds of general        formula 2    -   a) General procedure for the preparation of a neutral HMO        benzyl/substituted benzyl glycoside

A selected neutral HMO (1 equiv.) was dissolved/suspended in 1-10volumes (g/mL) of DMF, DMSO or a mixture thereof. The reaction mixturewas cooled to 0° C. and benzyl bromide/substituted benzyl bromide(1.2-1.4 equiv.) was added. A strong base such as sodium hydride,potassium hydride, calcium hydride, potassium t-butoxide, sodiumt-butoxide (1.2-1.4 equiv) was added at 0-40° C. and the reactionmixture was stirred for 6-24 hours at 0-60° C. Subsequently, water wasadded to quench the excess of base and the reaction mixture was stirredat RT for 30 minutes. The resulting reaction mixture was concentratedand purified in reverse phase chromatography, silica gel chromatography,ion-exchange chromatography, size-exclusion chromatography, etc. orcrystallized giving rise to the desired benzylated/substitutedbenzylated neutral HMO compound in 70-80% yields.

-   -   b) General procedure for the preparation of an acidic HMO        benzyl/substituted benzyl glycoside

A selected acidic HMO is dissolved in water and treated with the H⁺ formof an acidic ion-exchange resin, such as Amberlite IR-120, Dowex IL50,etc., to liberate the acidic HMO from its potential salt form. The ionexchange resin is filtered off and cesium carbonate was added to reachbasic pH, preferably pH 9-10. The resulting solution was lyophilized anddissolved/suspended in 1-10 volumes (g/mL) of DMF, DMSO or a mixturethereof. A methylating agent such as methyl iodide, methyl triflate,etc. in quantities related to the use of cesium carbonate (0.2-0.5equivalent excess) was added and the reaction mixture was stirred at0-60° C. for 4-24 hours. The reaction mixture was cooled to 0° C. andbenzyl bromide/substituted benzyl bromide in required quantities(1.2-1.5 equiv.) was added. Equivalent amount of strong base comparingto the benzylating/substituted benzylating agent used such as sodiumhydride, potassium hydride, calcium hydride, potassium t-butoxide,sodium t-butoxide was added at 0-40° C. and the reaction mixture wasstirred for 6-24 hours at 0-60° C. Subsequently, water was added tocreate an organic solvent:water ratio of 1:10 to 2:5 and the reactionmixture was stirred at 20-80° C. for 4-24 hours. The resulting reactionmixture was concentrated and purified in reverse phase chromatography,silica gel chromatography, ion-exchange chromatography, size-exclusionchromatography, etc. or crystallized or optionally converted into saltform giving rise to the desired benzylate/substituted benzylated acidicHMO compound in 60-70% yields.

-   -   c) General procedure for the preparation of a mixture of HMO        benzyl/substituted benzyl glycosides from a mixture comprising        at least one acidic HMO

The chemical composition of a mixture of HMOs was analyzed by LC-MS orany other suitable quantitative analytical method. Subsequently, the HMOmixture is dissolved in water and treated with the H⁺ form of an acidicion-exchange resin, such as Amberlite IR-120, Dowex IL50, etc., toliberate the acidic HMOs from their potential salt forms. The ionexchange resin is filtered off and cesium carbonate was added to reachbasic pH, preferably pH 9-10. The resulting solution was lyophilized anddissolved/suspended in 1-10 volumes (g/mL) of DMF, DMSO or a mixturethereof. A methylating agent such as methyl iodide, methyl triflate,etc. in quantities related to the use of cesium carbonate (0.2-0.5equivalent excess) was added and the reaction mixture was stirred at0-60° C. for 4-24 hours. The reaction mixture was cooled to 0° C. andbenzyl bromide/substituted benzyl bromide in required quantities(calculated according to LC-MS composition of the HMO mixture allowing0.2-0.5 equivalent excess) was added. An equivalent amount of strongbase comparing to the benzylating/substituted benzylating agent usedsuch as sodium hydride, potassium hydride, calcium hydride, potassiumt-butoxide, sodium t-butoxide was added at 0-40° C. and the reactionmixture was stirred for 6-24 hours at 0-60° C. Subsequently, water wasadded to create an organic solvent:water ratio of 1:10 to 2:5 and thereaction mixture was stirred at 20-80° C. for 4-24 hours. The resultingreaction mixture was concentrated and purified in reverse phasechromatography, silica gel chromatography, ion-exchange chromatography,size-exclusion chromatography, etc. giving the individualbenzylated/substituted benzylated compounds in 60-70% yields,respectively.

1-O-β-benzyl-LNnT

¹³C NMR (D₂O) δ: 105.6, 105.5, 105.4, 103.6 (anomeric carbons). Mp.:284-286° C.

1-O-β-(4-methylbenzyl)-LNnT

¹H NMR (D₂O): 7.3 (dd, 4H), 4.88 (d, 1H), 4.7 (m), 4.54 (d, 1H), 4.48(d, 1H), 4.42 (d, 1H), 4.34 (d), 4.0-3.5 (m), 3.34 (dd, 1H).

¹³C NMR (D₂O): 184.2, 177.6, 173.7, 141.5, 136.1, 131.9, 131.4, 105.6,105.5, 105.4, 103.6, 93.2, 84.7, 81.5, 81.0, 80.8, 78.0, 77.6, 77.4,77.1, 75.5, 75.2, 74.8, 74.0, 73.6, 72.9, 63.7, 62.8, 58.9, 56.4, 25.9,22.9.

1-O-β-(4-chlorobenzyl)-LNnT

¹H NMR (D₂O): 7.4 (s, 4H), 4.9 (d, 1H), 4.72 (m), 4.52 (d, 1H), 4.8 (d,1H), 4.42 (d, 1H), 4.16 (d, 1H), 4.0-3.52 (m).

¹³C NMR (D₂O): 138.9, 177.6, 138.3, 137.9, 136.2, 131.3, 105.6, 105.5,105.4, 103.7, 93.2, 86.1, 84.7, 81.5, 81.0, 80.8, 78.0, 77.5, 77.4,77.2, 77.1, 75.5, 75.2, 74.9, 63.7, 58.9, 57.8, 56.4, 24.8.

1-O-β-benzyl-LNT

¹H-NMR (D₂O, 400 MHz) δ 2.03 (s, 3H, CH ₃CONH), 3.35 (dd, 1H, J=8.18.5Hz, H-2), 3.49 (m, 1H, H-5″), 3.53 (m, H-2′″), 3.65 (m, 1H, H-3′″), 3.57(dd, 1H, J=8.1 9.0 Hz, H-4″), 3.58 (m, 1H, H-5), 3.59 (dd, 1H, J=7.710.0 Hz, H-2′), 3.62 (m, 1H, H-3), 3.63 (m, 1H, H-4), 3.71 (m, 1H,H-5′), 3.71 (m, 1H, H-5″), 3.73 (dd, 1H, J=3.310.0 Hz, H-3′), 3.76 (m,2H, H-6ab′″), 3.76 (m, 2H, H-6ab′), 3.80 (m, 1H, H-6a″), 3.80 (dd, 1H,J=5.012.2 Hz, H-6a), 3.82 (dd, 1H, J=8.110.5 Hz, H-3″), 3.90 (m, 1H,H-613″), 3.90 (dd, 1H, J=8.4 10.5 Hz, H-2″), 3.92 (d, 1H, J=3.3 Hz,H-4″), 3.98 (dd, 1H, J=1.6 12.2 Hz, H-6b), 4.15 (d, 1H, J=3.3 Hz, H-4′),4.44 (d, 1H, J=7.7 Hz, H-1′), 4.45 (d, 1H, J=7.7 Hz, H-1′″), 4.56 (d,1H, J=8.1 Hz, H-1), 4.73 (d, 1H, J=8.4 Hz, H-1″), 4.76 (d, 1H, J=11.7Hz, CH ₂Ph), 4.94 (d, 1H, J=11.7 Hz, CH ₂Ph), 7.40-7.50 (m, 5H, Ph).

¹³C-NMR (D₂O, 100 MHz) δ 24.9 (CH₃CONH), 57.4 (C-2″), 62.8 (C-6), 63.2(C-6″), 63.7 (C-6′″), 63.7 (C-6′), 71.0 (C-4′), 71.2 (C-4′″), 71.3(C-4″), 72.7 (C-2′), 73.4 (C-2′″), 74.2 (CH₂Ph), 75.2 (C-3′″), 75.5(C-2), 77.1 (C-3), 77.5 (C-5′), 77.6 (C-5′″), 77.9 (C-5), 78.0 (C-5″),81.1 (C-4), 84.7 (C-3′), 84.8 (C-3″), 103.7 (C-1), 105.3 (C-1″), 105.6(C-1′), 106.2 (C-1′″), 131.1 (Ph), 131.4 (2C, Ph), 131.5 (2C, Ph), 139.2(Ph), 177.7 (CH₃ CONH).

M.p. 245° C. (dec.). [α]_(D) ²²=−10.3 (c=1, H₂O).

1-O-β-(4-methylbenzyl)-LNT

¹H-NMR (D₂O, 300 MHz) δ 1.97 (s, 3H), 2.29 (s, 3H), 3.27 (dd, 1H,J=8.18.5 Hz), 3.39-3.87 (m, 21H), 3.92 (dd, 1H, J=1.812.3 Hz), 4.09 (d,1H, J=3.3 Hz), 4.37 (d, 1H, J=8.1 Hz), 4.38 (d, 1H, J=7.8 Hz), 4.47 (d,1H, J=8.1 Hz), 4.65 (d, 1H, J=11.7 Hz), 4.67 (d, 1H, J=8.1 Hz), 4.83 (d,1H, J=11.7 Hz), 7.22 (d, 2H, J=8.1 Hz), 7.30 (d, 2H, J=8.1 Hz).

¹³C-NMR (D₂O, δ5.4 MHz) δ 23.1, 25.0, 57.7, 62.8, 63.2, 63.7, 63.8,71.0, 71.1, 71.3, 72.7, 73.4, 74.1, 75.2, 75.5, 77.1, 77.5, 77.6, 77.9,78.0, 81.1, 84.7, 84.8, 103.6, 105.3, 105.7, 106.2, 131.7 (2C), 132.0(2C), 136.2, 141.5, 177.7.

1-O-β-benzyl-6′-O-sialyl-lactose

¹H-NMR (CD₃OD) δ (ppm): 1.63 (t, 1H, J=11.9 Hz); 2.00 (s, 3H); 2.78 (dd,1H, J=4.5 Hz, J=12.2 Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H);3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5 Hz, J=10 Hz); 4.35 (m, 1H); 4.42(d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz); 7.22-7.37 (m, 3H); 7.38-7.46(m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.55, 41.26, 52.70, 60.81, 63.21, 63.42,68.56, 69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55, 73.78, 74.47,75.28, 75.32, 80.03, 100.29, 101.89, 103.36, 127.36, 127.87, 128.01,128.12, 137.72, 173.36, 173.88.

1-O-β-benzyl-6′-O-sialyl-lactose sodium salt

¹H-NMR (CD₃OD) δ (ppm): 1.63 (t, 1H, J=11.9 Hz); 2.02 (s, 3H); 2.82 (m,1H); 3.33-3.45 (m, 4H); 3.46-3.61 (m, 5H); 3.62-3.97 (m, 12H); 4.02 (dd,1H, J=7.5 Hz, J=10 Hz); 4.37 (m, 2H); 4.66 (d, 1H, J=11.9 Hz); 7.31 (m,3H); 7.41 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.10, 40.60, 52.04, 60.22, 62.78, 67.90,68.51, 68.69, 70.11, 70.61, 70.64, 71.44, 72.44, 72.83, 73.17, 73.92,74.50, 74.70, 79.82, 99.76, 101.23, 103.52, 127.00, 127.44, 127.56,137.21, 172.68, 173.28.

1-O-β-benzyl-6′-O-sialyl-lactose zinc salt

¹H-NMR (CD₃OD) δ (ppm): 1.71 (t, 1H, J=11.1 Hz); 2.00 (s, 3H); 2.78 (dd,1H, J=4.5 Hz, J=12.2 Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H);3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5 Hz, J=10 Hz); 4.35 (m, 1H); 4.42(d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz); 7.22-7.37 (m, 3H); 7.38-7.46(m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.77, 41.09, 52.78, 60.87, 61.32, 63.06,63.64, 63.21, 68.26, 69.11, 70.67, 71.22, 71.36, 72.01, 73.00, 73.37,73.58, 73.66, 74.39, 75.19, 75.32, 75.87, 79.40, 80.35, 99.92, 101.86,101.94, 103.86, 104.00, 127.55, 127.99, 128.13, 137.83, 174.03, 174.08.

1-O-β-benzyl-6′-O-sialyl-lactose ethanolammonium salt

¹H-NMR (CD₃OD) δ (ppm): 1.63 (dd, 1H, J=11.7 Hz, J=12.2 Hz); 2.00 (s,3H); 2.78 (dd, 1H, J=4.5 Hz, J=12.2 Hz); 2.92 (m, 4H); 3.33-3.47 (m,2H); 3.46-3.57 (m, 5H); 3.58-3.78 (m, 9H); 3.78-3.90 (m, 5H); 3.93 (dd,1H, J=2.5, J=11.7 Hz); 4.02 (dd, 1H, J=7.5 Hz, J=10 Hz); 4.35 (m, 1H);4.42 (d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz); 7.22-7.37 (m, 3H);7.38-7.46 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.55, 41.26, 44.83, 52.70, 58.85, 60.81,63.21, 63.42, 68.56, 69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55,73.78, 74.47, 75.28, 75.32, 80.03, 99.98, 100.89, 102.36, 127.36,127.87, 128.01, 128.12, 137.72, 173.36, 173.88.

1-O-β-benzyl-6′-O-sialyl-lactose tris-(hydroxymethyl)-methyl ammoniumsalt

¹H-NMR (CD₃OD) δ (ppm): 1.63 (dd, 1H, J=11.9 Hz, J=12.1 Hz); 2.00 (s,3H); 2.78 (dd, 1H, J=4.5 Hz, J=12.2 Hz); 3.33-3.48 (m, 2H); 3.48-3.79(m, 19H); 3.93 (dd, 1H, J=2.5, J=11.8 Hz); 4.02 (dd, 1H, J=7.5 Hz, J=10Hz); 4.35 (m, 1H); 4.42 (d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz);7.22-7.37 (m, 3H); 7.38-7.46 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.60, 41.22, 52.71, 59.62, 60.89, 61.38,63.23, 63.39, 68.64, 68.97, 69.25, 70.64, 71.26, 73.68, 75.24, 75.27,80.16, 100.39, 101.87, 103.94, 127.52, 127.97, 128.00, 128.11, 137.85,173.40, 173.90.

1-O-β-benzyl-6′-O-sialyl-lactose diethyl ammonium salt

¹H-NMR (CD₃OD) δ (ppm): 1.28 (t, 6H, J=11.8 Hz); 1.65 (dd, 1H, J=11.9Hz, J=12.1 Hz); 2.00 (s, 3H); 2.79 (dd, 1H, J=4.5 Hz, J=12.2 Hz); 3.00(q, 4H); 3.33-3.57 (m, 7H); 3.57-3.77 (m, 5H); 3.78-3.89 (m, 4H); 3.93(dd, 1H, J=11.9 Hz, J=2.4 Hz); 4.00 (dd, 1H, J=7.1 Hz, J=9.7 Hz); 4.35(m, 1H); 4.42 (d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.8 Hz); 7.22-7.37 (m,3H); 7.38-7.46 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 10.64, 21.57, 41.29, 42.29, 52.74, 60.89,63.23, 63.43, 68.63, 68.95, 69.30, 70.62, 71.25, 72.04, 73.06, 73.41,73.73, 74.47, 75.26, 75.28, 80.15, 100.41, 101.90, 103.95, 127.51,127.97, 127.99, 128.10, 137.88, 173.35, 173.85.

1-O-β-benzyl-6′-O-sialyl-lactose choline salt

¹H-NMR (CD₃OD) δ (ppm): 1.63 (dd, 1H, J=11.9 Hz, J=12.1 Hz); 1.98 (s,3H); 2.80 (dd, 1H, J=4.5 Hz, J=12.2 Hz); 3.19 (s, 9H); 3.33-3.56 (m,8H); 3.56-3.77 (m, 7H); 3.78-3.94 (m, 5H); 3.95-4.04 (m, 4H); 4.34 (m,1H); 4.39 (d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.8 Hz); 7.22-7.36 (m, 3H);7.40 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.56, 40.95, 41.34, 52.75, 53.48, 53.53,53.59, 55.92, 60.96, 63.15, 63.50, 63.84, 67.85, 68.65, 68.90, 69.31,69.47, 70.61, 71.28, 72.05, 73.05, 73.41, 73.72, 74.45, 75.29, 76.89,80.17, 100.42, 101.92, 103.97, 109.98 127.52, 127.97, 128.11, 137.90,138.29, 173.26, 173.85.

1-O-β-(4-chlorobenzyl)-6′-O-sialyl-lactose

¹H-NMR (CD₃OD) δ (ppm): 1.62 (t, 1H, J=12 Hz); 2.00 (s, 3H); 2.77 (dd,1H, J=4.7 Hz, J=12.1 Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H);3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5 Hz, J=10 Hz); 4.35 (m, 1H); 4.42(d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz); 7.24 (m, 2H); 7.32 (m, 2H).

¹³C-NMR (CD₃OD) δ (ppm): 21.52, 41.23, 52.72, 60.79, 63.23, 63.42,68.61, 69.03, 69.31, 70.56, 71.14, 72.13, 73.11, 73.56, 73.79, 74.48,75.23, 75.31, 80.09, 100.17, 101.91, 103.21, 127.36, 127.89, 133.01,136.76, 173.29, 173.78.

1-O-β-(4-methylbenzyl)-6′-O-sialyl-lactose

¹H-NMR (CD₃OD) δ (ppm): 1.63 (t, 1H, J=11.9 Hz); 2.00 (s, 3H); 2.32 (s,3H); 2.78 (dd, 1H, J=4.5 Hz, J=12.2 Hz); 3.28-3.49 (m, 4H); 3.50-3.79(m, 9H); 3.80-3.97 (m, 5H); 4.02 (dd, 1H, J=7.5 Hz, J=10 Hz); 4.35 (m,1H); 4.42 (d, 1H, J=7.8 Hz); 4.66 (d, 1H, J=11.7 Hz); 7.21 (m, 4H).

¹³C-NMR (CD₃OD) δ (ppm): 20.02, 21.55, 41.26, 52.70, 60.81, 63.21,63.42, 68.56, 69.01, 69.30, 70.66, 71.15, 72.12, 73.08, 73.55, 73.78,74.47, 75.28, 75.32, 80.03, 99.98, 100.89, 102.36, 127.36, 127.87,128.01, 128.12, 133.26, 137.72, 173.36, 173.88.

1-O-β-(1-naphthylmethyl)-6′-O-sialyl-lactose

¹H (CD₃OD) δ (ppm): 1.62 (t, 1H, J=11.8 Hz); 2.00 (s, 3H); 2.78 (dd, 1H,J=4.5 Hz, J=12.2 Hz); 3.28-3.49 (m, 4H); 3.50-3.79 (m, 9H); 3.80-3.97(m, 5H); 4.02 (dd, 1H, J=7.6 Hz, J=10.1 Hz); 4.35 (m, 1H); 4.42 (d, 1H,J=7.8 Hz); 4.68 (d, 1H, J=11.8 Hz); 7.32-7.54 (m, 4H); 7.7 (m, 2H); 7.95(m, 1H).

¹³C (CD₃OD) δ (ppm): 21.54, 41.28, 52.72, 60.81, 63.20, 63.44, 68.53,69.06, 69.38, 70.64, 71.12, 72.14, 73.05, 73.54, 73.65, 74.45, 75.24,75.33, 80.02, 100.25, 101.87, 103.21, 122.91, 124.82, 125.25, 125.93,127.06, 128.72, 129.16, 132.80, 133.12, 135.97, 136.88, 173.36, 173.88.

1-O-β-benzyl-3′-O-sialyl-lactose

¹H-NMR (500 MHz, D₂O): δ[ppm]=7.46-7.42 (m, 4H, H_((a/b)arom)); 4.91 (d,1H, CH₂a-Bn); 4.74 (d, 1H, CH₂b-Bn); 4.55-4.52 (m, 2H, H-1/H-1′); 4.11(dd, 1H, H-3′); 2.76 (dd, 1H, H-3″_(eq)); 2.04 (s, 3H, COCH₃); 1.81 (dd,1H, H-3″_(ax)).

J_((a,b)-Bn)=11.8; J_(2′,3′)=9.9; J_(3′,4′)=2.9; J_(3″ax,3″eq)=12.4;J_(3″ax,4″)=12.1; J_(3″eq,4″)=4.5 Hz.

¹³C-NMR (126 MHz, D₂O): 6 [ppm]=135.2, 133.5 (quart. C_(arom)); 130.2(CH_(a-arom)); 128.6 (CH_(b-arom))_(;) 102.6 (C-1′); 101.1 (C-1); 51.7(C-5″); 39.6 (C-3″); 22.0 (COCH₃).

1-O-β-(4-methylbenzyl)-3′-O-sialyl-lactose

¹H-NMR (500 MHz, D₂O): δ [ppm]=7.36 (d, 2H, H-a_(arom)); 7.28 (d, 2H,H-b_(arom)); 4.89 (d, 1H, CH₂a-Bn); 4.72 (d, 1H, CH₂b-Bn); 4.54-4.52 (m,2H, H-1/H-1′); 4.12 (dd, 1H, H-3′); 2.76 (dd, 1H, H-3″_(eq)); 2.35 (s,3H, CH₃-Tol); 2.04 (s, 3H, COCH₃), 1.81 (dd, 1H, H-3″_(ax)).

J_((a,b)arom)=7.9; J_((a,b)-Bn)=11.5; J_(2′,3′)=9.9; J_(3′,4′)=3.0;J_(3″ax,3″eq)=12.5; J_(3″,4″)=12.2; J_(3″eq,4″)=4.6 Hz.

¹³C-NMR (126 MHz, D₂O): δ [ppm]=138.8, 133.4 (quart. C_(arom)); 129.3(CH_(a-arom)); 129.0 (CH_(b-arom))_(;) 102.6 (C-1′); 100.9 (C-1); 99.8(C-2″), 51.7 (C-5″); 39.6 (C-3″); 22.0 (COCH₃); 20.2 (CH₃-Tol).

1-O-β-(2-naphthylmethyl)-3′-O-sialyl-lactose

¹H-NMR (500 MHz, D₂O): 6 [ppm]=7.99-7.97 (m, 4H, H-arom); 7.62-7.59 (m,3H, H-arom); 5.10 (d, 1H, CH₂a-Bn); 4.95 (d, 1H, CH₂b-Bn); 4.60 (d, 1H,H-1); 4.53 (d, 1H, H-1′); 4.12 (dd, 1H, H-3′); 2.77 (dd, 1H, H-3″_(eq));2.04 (s, 3H, COCH₃); 1.81 (dd, 1H, H-3″_(ax)).

J_((a,b)-Bn)=11.9; J_(1,2)=8.0; J_(1′,2′)=7.9; J_(2′,3′)=9.9;J_(3′,4′)=3.0; J_(3″ax,3″eq)=12.5; J_(3″ax,4″)=12.1; J_(3″eq,4″)=4.6 Hz.

¹³C-NMR (126 MHz, D₂O): 6 [ppm]=128.3, 127.9, 127.7, 127.6, 126.6, 126.5(CH_(arom)); 102.6 (C-1′); 101.1 (C-1); 51.7 (C-5″); 22.0 (COCH₃).

1-O-β-(3-phenylbenzyl)-3′-O-sialyl-lactose

¹H-NMR (400 MHz, D₂O): 6 [ppm]=7.75-7.44 (m, 9H, H-arom); 4.99 (d, 1H,CH₂a-Bn); 4.82 (d, 1H, CH₂b-Bn); 4.57 (d, 1H, H-1); 4.53 (d, 1H, H-1′);4.13 (dd, 1H, H-3′); 2.78 (dd, 1H, H-3″_(eq)); 2.05 (s, 3H, COCH₃); 1.82(dd, 1H, H-3″_(ax)).

J_((a,b)-Bn)=11.8; J_(1,2)=8.0; J_(1′,2′)=7.9; J_(2′,3′)=9.9,J_(3′,4′)=3.1; J_(3″ax,3″eq)=12.5; J_(3″ax,4)=12.0; J_(3″eq,4″)=4.6 Hz.

¹³C-NMR (100 MHz, D₂O): δ [ppm]=140.9, 140.3, 137.5 (quart. C_(arom));129.4, 129.2, 127.9, 127.8, 127.1, 127.0, 126.9, (CH_(arom)); 102.7(C-1′); 101.2 (C-1); 99.6 (C-2″); 51.8 (C-5″); 39.7 (C-3″); 22.1(COCH₃).

1-O—O-benzyl-2′-O-fucosyl-lactose

Characteristic ¹H NMR peaks (DMSO-d₆) δ: 7.41-7.25 (m, 5H, aromatic),5.20 (d, 1H, J_(1″,2″)=2 Hz, H-1″), 4.82 and 4.59 (ABq, 2H, J_(gem)=12.3Hz, —CH₂Ph), 4.32 (d, 1H, J_(1′,2′)=7.31 Hz, H-1′), 4.28 (d, 1H,J_(1,2)=7.79 Hz, H-1), 1.05 (d, 1H, J_(5″,6″)=6.43 Hz, H-6″).

¹³C NMR (DMSO-d₆) δ: 137.97, 128.15, 128.15, 127.58, 127.58 and 127.43(aromatic), 101.86, 100.94 and 100.20 (C-1, C-1′ and C-1″), 77.68,76.78, 75.36, 75.33, 74.70, 73.79, 73.45, 71.60, 69.72, 69.69, 68.74,68.20, 66.38, 60.23 and 59.81 (C-2, C-3, C-4, C-5, C-6, C-2′, C-3′,C-4′, C-5′, C-6′, C-2″, C-3″, C-4″, C-5″ and CH₂Ph), 16.47 (C-6″).

1-O-β-(4-nitrobenzyl)-2′-O-fucosyl-lactose

Characteristic ¹H-NMR peaks (DMSO-d₆) δ: 8.20 and 7.68 (each d, 4H,aromatic), 5.04 (d, 1H, J_(1″,2″)=2 Hz, H-1″), 4.97 and 4.76 (ABq, 2H,J_(gem)=12.3 Hz, —CH₂Ph), 4.40 (d, 1H, J_(1′,2′)=9.53 Hz, H-1′), 4.32(d, 1H, J_(1,2)=8.04 Hz, H-1), 1.04 (d, 1H, J_(5″,6″)=6.43 Hz, H-6″).

¹³C-NMR (DMSO-d₆) δ: 162.38, 147.68, 127.95, 127.95, 123.33 and 123.33(aromatic), 102.18, 100.95 and 100.18 (C-1, C-1′ and C-1″), 77.62,76.74, 75.36, 75.36, 74.61, 73.78, 73.45, 71.59, 69.67, 68.74, 68.67,68.21, 66.38, 60.22 and 59.77 (C-2, C-3, C-4, C-5, C-6, C-2′, C-3′,C-4′, C-5′, C-6′, C-2″, C-3″, C-4″, C-5″ and CH₂Ph), 16.45 (C-6″).

2. Hydrogenolysis of compounds of general formula 2

a) 40 g (50.1 mmol) of 1-O-β-benzyl-LNnT were dissolved in 200 ml ofwater, 1.6 g of Pd—C and 400 μl of acetic acid was added, and themixture was stirred at rt. under H₂-atmosphere (approx. 40 bars) for 2days. The catalyst was filtered off, the cake was washed with water, andthe filtrate was added dropwise to 1.61 of acetone, then chilled,filtered and the collected solid was dried under vacuum to yield 31.5 gof white powder of LNnT (44.5 mmol, 89%).

b) 1-O-benzyl-β-LNT (5 g, 6.27 mmol) was suspended in water (20 mL) andthe pH was adjusted to 5.8 by addition of 1M aq. HCl. Palladium oncharcoal (0.5 g, 10%) was added and the reaction flask was evacuated andthen saturated with H₂ (4 bar). The reaction temperature was set to 50°C. and after stirring for 1.5 hour the temperature was allowed to reachRT, the catalyst was removed by filtration and water was used forwashing (10 mL). The filtrate was concentrated to dryness and 3.46 g(78%) of white solid LNT was obtained.

c) To a solution of 40 g of 1-O-β-benzyl-6′-O-sialyl-lactose in amixture of methanol and water (250 mL+300 mL) 4 g of Pd/C (10%) wereadded. The reaction mixture was stirred 2 d at RT under H₂ pressure(balloon). The mixture was then filtered through a pad of Celite and thesolvent was evaporated in vacuo. The residue was dissolved in 80 mL ofH₂O and dropped to 1200 mL of EtOH. The slurry was filtrated, the solidwas washed with EtOH, acetone and a mixture of 1/1 acetone/Et₂O. Thesolid was dried to give 35 g of 6′-O-sialyllactose.

d) To a solution of 1-O-β-benzyl-2′-O-fucosyl-lactose (5.0 g) inmethanol (50 ml) 100 mg 10% palladium on charcoal is added. Thesuspension is stirred under hydrogen atmosphere at rt for 4 hours. Thecatalyst is filtered off, washed with water and the filtrate isevaporated to yield 2′-O-fucosyl-lactose as amorphous white solid: 4.2g.

1. A method for purifying, separating and/or isolating anoligosaccharide of general formula 1 or a salt thereof

wherein R₁ is fucosyl or H, R₂ is fucosyl or H, R₃ is selected from H,sialyl, N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein theN-acetyl lactosaminyl group may carry a glycosyl residue comprising oneor more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups;each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can besubstituted with one or more sialyl and/or fucosyl residue, R₄ isselected from H, or sialyl and N-acetyl-lactosaminyl groups optionallysubstituted with a glycosyl residue comprising one or moreN-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each ofthe N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substitutedwith one or more sialyl and/or fucosyl residue, wherein at least one ofthe R₁, R₂, R₃ or R₄ groups differs from H, comprising: a) subjectingone or more compounds of general formula 1 to an anomeric O-alkylationreaction in the presence of R—X to yield a mixture comprising one ormore compounds of general formula 2 or salts thereof.

wherein X is a leaving group such as halogen, alkyl- or arylsulfonyloxy,R is a group removable by hydrogenolysis, and R₁, R₂, R₃ and R₄ are asdefined above, and wherein at least one of the R₁, R₂, R₃ or R₄ groupsdiffers from H, b) subjecting the mixture comprising one or morecompounds of general formula 2 obtained in step a) to chromatographyand/or crystallization to give one or more individual compounds ofgeneral formula 2 each in substantially pure form, c) subjecting anindividual compound of general formula 2 in substantially pure formobtained in step b) to catalytic hydrogenolysis to yield a compound ofgeneral formula
 1. 2. The method according to claim 1, wherein group Rin compound R—X and compounds of general formula 2 is benzyl or 1- or2-naphthylmethyl optionally substituted with one or more groups selectedfrom phenyl, alkyl or halogen.
 3. The method according to claim 1,wherein the chromatography is reverse phase or size exclusionchromatography.
 4. The method according to claim 1, wherein compounds ofgeneral formula 1 are represented by general formulae 1a, 1b or 1c,

and compounds of general formula 2 are represented by general formulae2a, 2b or 2c

herein R, R₁ and R₂ are as defined in claim 1, R_(3a) is anN-acetyl-lactosaminyl group optionally substituted with a glycosylresidue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosylgroup; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups canbe substituted with one or more sialyl and/or fucosyl residue, R_(4a) isH or an N-acetyl-lactosaminyl group optionally substituted with alacto-N-biosyl group; each of the N-acetyl-lactosaminyl andlacto-N-biosyl groups can be substituted with one or more sialyl and/orfucosyl residue, R_(3b) is a lacto-N-biosyl group optionally substitutedwith one or more sialyl and/or fucosyl residue(s), R_(4b) is H or anN-acetyl-lactosaminyl group optionally substituted with one or twoN-acetyl-lactosaminyl and/or one lacto-N-biosyl groups; each of theN-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted withone or more sialyl and/or fucosyl residues, R₅ is independently H orsialyl, wherein at least one of R₁, R₂ or R₅ differs from H.
 5. Themethod according to claim 1, wherein compounds of general formula 1a or2a are selected from the group consisting of lacto-N-neotetraose,para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose,para-lacto-N-octaose and lacto-N-neooctaose derivatives optionallysubstituted with one or more sialyl and/or fucosyl residue, or salts ofthese compounds, and compounds of general formula 1b or 2b are selectedfrom the group consisting of lacto-N-tetraose, lacto-N-hexaose,lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose andlacto-N-neodecaose derivatives optionally substituted with one or moresialyl and/or fucosyl residue, or salts of these compounds.
 6. Themethod according to claim 4, wherein compounds of general formula 1a,1b, 1c, 2a, 2b or 2c are selected from the group consisting of2′-O-fucosyllactose, 3-O-fucosyllactose, 2′,3-di-O-fucosyllactose,3′-O-sialyllactose, 6′-O-sialyllactose, 3′-O-sialyl-3-O-fucosyllactose,lacto-N-tetraose, lacto-N-neotetraose,Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNFP-I), Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (LNFP-II), Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc(LNFP-III), Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-4)Glc (LNFP-V),Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LST-a),Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (LST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LST-c),Neu5Acα2-3Galβ1-3(Fucα1-4) GlcNAcβ1-3Galβ1-4Glc (FLST-a),Fucα1-2Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (FLST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FLST-c),Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-I),Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-III),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (DS-LNT),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FDS-LNT I)and Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FDS-LNTII), or salts thereof, for general formulae 1a, 1b or 1c, or theirrespective R-glycosides or salts thereof for general formulae 2a, 2b or2c.
 7. Compounds of general formula 2′ or salts thereof

wherein R is a group removable by hydrogenolysis, R₁ is fucosyl or H, R₂is fucosyl or H, R₃ is selected from H, sialyl, N-acetyl-lactosaminyland lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group maycarry a glycosyl residue comprising one or more N-acetyl-lactosaminyland/or one or more lacto-N-biosyl groups; each of theN-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted withone or more sialyl and/or fucosyl residue, R₄ is selected from H, sialyland N-acetyl-lactosaminyl groups optionally substituted with a glycosylresidue comprising one or more N-acetyl-lactosaminyl and/or one or morelacto-N-biosyl groups; each of the N-acetyl-lactosaminyl andlacto-N-biosyl groups can be substituted with one or more sialyl and/orfucosyl residue, wherein at least one of the R₁, R₂, R₃ or R₄ groupsdiffers from H, and provided that the following compounds are excluded:R-glycosides of LNnT, 1-O-β-benzyl-LNT, R-glycosides of6′-O-sialyl-lactose and salts thereof, 1-O-β-benzyl-3′-O-sialyl-lactoseNa salt, 1-O-β-(4,5-dimethoxy-2-nitro)-benzyl-3′-O-sialyl-lactose Nasalt.
 8. A compound according to claim 7, wherein compounds of generalformula 2′ are characterized by general formulae 2′a, 2′b or 2′c orsalts thereof

wherein R, R₁ and R₂ are as defined in claim 7, R_(3a) is anN-acetyl-lactosaminyl group optionally substituted with a glycosylresidue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosylgroup; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups canbe substituted with one or more sialyl and/or fucosyl residue, R_(4a) isH or an N-acetyl-lactosaminyl group optionally substituted with alacto-N-biosyl group; each of the N-acetyl-lactosaminyl andlacto-N-biosyl groups can be substituted with one or more sialyl and/orfucosyl residue, R_(3b) is a lacto-N-biosyl group optionally substitutedwith one or more sialyl and/or fucosyl residue, R_(4b) is H or anN-acetyl-lactosaminyl group optionally substituted with one or twoN-acetyl-lactosaminyl and/or one lacto-N-biosyl groups; each of theN-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted withone or more sialyl and/or fucosyl residue, R₅ is independently H orsialyl, wherein at least one of R₁, R₂ or R₅ differs from H.
 9. Acompound according to claim 8, wherein general formula 2′a represents acompound selected from the group consisting of lacto-N-neotetraose,para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose,para-lacto-N-octaose and lacto-N-neooctaose R-glycosides optionallysubstituted with one or more sialyl and/or fucosyl residue, or saltsthereof, and general formula 2′b represents a compound selected from thegroup consisting of lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose,iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose R-glycosidesoptionally substituted with one or more sialyl and/or fucosyl residue,or salts thereof.
 10. A compound according to claim 7, which is anR-glycoside of a compound selected from the group consisting of2′-O-fucosyllactose, 3-O-fucosyllactose, 2′,3-di-O-fucosyllactose,3′-O-sialyllactose, 6′-O-sialyllactose, 3′-O-sialyl-3-O-fucosyllactose,lacto-N-tetraose, lacto-N-neotetraose,Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNFP-I),Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (LNFP-II),Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcGalβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-4)Glc (LNFP-V),Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LST-a),Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (LST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LST-c),Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FLST-a),Fucα1-2Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (FLST-b),Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FLST-c),Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-I),Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-II),Galβ-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (LNDFH-III),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (DS-LNT),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FDS-LNT I)and Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FDS-LNTII), or salts thereof.